Growth and Stability of Thrombi in Flowing Citrated Blood: Assessment of Platelet-Surface Interactions with Computer-Assisted Morphometry

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Abstract
The differential quantitation of platelet deposition in perfusion studies is a major problem. We report on methods to prepare semithin sections of platelet deposits on collagen coated on glass and plastic cover slips, to study growth and stability of thrombi in three dimensions, and the development of a computer-assisted differential quantitation of platelet-collagen interactions. The interactions were quantified as percentage of the surface covered with platelets (platelet adhesion), thrombus height, thrombus density and thrombus area per unit sectional length, respectively. Cover slips coated with fibrillar equine collagen in parallelplate perfusion chambers were exposed to flowing citrated blood at shear rates ranging from 200 to 2,600 s−1. Thrombi, partially enmeshed in the collagen meshwork, prevailed on the surface at all shear rates. Maximal platelet adhesion and thrombus density were seen at >5 μg/cm2 collagen, while thrombus area and height were maximal at >10 μg/cm2. The volume of the thrombi appeared correlated to the number of deposited platelets (r = 0.92). En face preparations showed deposits of platelet islands which grew in diameter with time, particularly in the direction of the blood flow, becoming progressively confluent. Sections cut parallel to the direction of the blood stream indicated that this growth pattern was at least partially caused by thrombi bent in the direction of the blood flow. This view is consistent with data from corresponding sections cut perpendicular to the direction of the blood flow showing that the initial thrombus growth at 2 min is isotropic, while anisotropic growth, characterized with decreased growth in height, is observed at 5 and 10 min. Our three-dimensional analysis suggests that the growth occurs mainly in height, and that blood shear forces may bend the thrombi toward the surface resulting in platelet thrombi preferentially elongated in the direction of the blood flow.

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